FIGURE 6.
Monitoring UPS function in zebrafish embryos. A, we created a system involving transient expression of a UPS fluorescent reporter. A polyubiquitination signal was joined to an otherwise stable YFP to create the Ub-R-YFP fusion protein. Ub-R-YFP was left undegraded after CDC48 knockdown and was degraded after CDC48 overexpression. B, fluorescent proteins were detected by Western blotting using the anti-GFP antibody. Ub-R-YFP, YFP-CL1, UbG76V-YFP, and CD3δ-YFP were introduced in the presence of the cont-MO, zCDC48-MO, or zCDC48 constructs. C, immunohistochemistry of ubiquitinated fluorescent proteins and zCDC48 in brain is shown. GFP and Ub-R-YFP were injected and stained by anti-GFP and CDC48. The numbers of zCDC48- and GFP-positive cells were counted.